Month: September 2013

According to the Center for Disease Control, stroke is a leading cause of death in the United States. Fortunately stroke has been the subject of significant research efforts, but unfortunately, developing treatments that ensure complete recovery for stroke patients is extremely challenging. The challenge increase when more than a few hours have passed between onset of the stroke and administration of treatment.

Thus a new study released in STEM CELLS Translational Medicine has generated more than a little excitement. This study indicates that indicates that endothelial precursor cells (EPCs), which are found in the bone marrow, umbilical cord blood, and rarely in peripheral blood, can make a significant difference for these patients’ recovery. The contribution of EPCs even extends to the later stages of stroke. In animal studies, EPC implantation into the brain after a stroke minimized the initial brain injury and helped repair the stroke damage.

“Previous studies indicated that stem/progenitor cells derived from human umbilical cord blood (hUCB) improved functional recovery in stroke models,” noted Branislava Janic, Ph.D., a member of Henry Ford Health System’s Cellular and Molecular Imaging Laboratory in Detroit and lead author of the study. “We wanted to examine the effect of hUCB-derived AC133+ endothelial progenitor cells (EPCs) on stroke development and resolution in rats.”

Dr. Janic and his team injected EPCs into the brains of rats that had suffered strokes. When they later examined the animals using MRI, they found that the transplanted EPCs had selectively migrated to the injured area, stopped the tissue damage from spreading, initiated regeneration, and affected the time course for stroke resolution. The lesion size in the brain was significantly decreased at a dose of 10 million cells, if the cells were given as early as seven days after the onset of the stroke.

“This led us to conclude that cord blood-derived EPCs can significantly contribute to developing more effective treatments that allow broader time period for intervention, minimize the initial brain injury and help repair the damage in later post-stroke phases,” Dr. Janic said.

“The early signs of stroke are often unrecognized, and many patients cannot take advantage of clot-busting treatments within the required few hours after stroke onset,” said Anthony Atala, M.D., editor of STEM CELLS Translational Medicine and director of the Wake Forest Institute for Regenerative Medicine. “In this animal study, a combination of stem cells shows promise for healing stroke damage when administered 24 hours after the stroke.”

Embryonic stem cells have the capacity to differentiate into every cell in the adult body. One cell type into which embryonic stem cells (ESCs) can be differentiated rather efficiently is cardiomyocytes, which is a fancy term for heart muscle cells. The protocol for making heart muscle cells from ESCs is well worked out, and the conversion is rather efficient and the purification schemes that have been developed are also rather effective (for example, see Cao N, et al., Highly efficient induction and long-term maintenance of multipotent cardiovascular progenitors from human pluripotent stem cells under defined conditions. Cell Res. 2013 Sep;23(9):1119-32. doi: 10.1038/cr.2013.102 and Mummery CL et al., Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview. Circ Res. 2012 Jul 20;111(3):344-58).

Using these cells in a clinical setting has two large challenges. The first is that embryonic stem cell derivatives are rejected by the immune system of the recipient, thus setting up the patient for a graft versus host response to the implanted tissue, thus making the patient even sicker than when they started. The second problem is that heart muscle cells made from ESCs are immature and cause the heart to beat abnormally fast thus causing “tachyarrythmias” and died within the first two weeks after the transplant (see Liao SY, et al., Heart Rhythm 2010 7:1852-1859).

Both of these problems are large problems, but the laboratory of Ronald Li at the University of Hong Kong at used a genetic engineering trick to make heart muscle cells from mouse embryonic stem cells to seemingly fix this problem.

Li and his colleagues engineered mouse ESCs with a gene for a potassium rectifier channel that could be induced with drugs. Then they differentiated these genetically ESCs into heart muscle cells. This potassium rectifier channel (Kir2.1) is not present in immature heart muscle cells and putting it into these cells might cause them to beat at a slower rate.

These engineered ESC-derived heart muscle cells were tested for their electrophysiological properties first. Without the drug that induces KIR2.1, the heart muscle cells showed very abnormal electrical properties. However, once the drug was added, their electrical properties looked much more normal.

Then they induced heart attacks in laboratory animals and implanted their engineered ESC-derived heart muscle cells 1 hour after the heart attacks were induced. Animals not given the drug to induce the expression of Kir2.1 faired very poorly and had episodes of tachyarrythmia (really fast heart beat) and over half of them died by 5 weeks after the implantation. Essentially the implanted animals did worse than those animals that had had a heart attack that were not treated. However, those animals that were given the drug that induces the expression of Kir2.1 in heart muscle cells did much better. The survival rate of these animals was higher than the untreated animals after about 7 weeks after the procedure. Survival rates increased by only a little, but the increase was significant. Also, the animals that died did not die of tachyarrythmias. In fact the rate of tachyarrythmias in the animals given the inducing drug (which was doxycycline by the way) had significantly lower levels of tachyarrythmia than the other two groups.

Other heart functions were also significantly affected. The ejection fraction in the animals that ha received the Kir2.1-expression heart muscle cells was 10-20% higher than the control animals. Also the density of blood vessels was substantially higher in both sets of animals treated with ESC-derived heart muscle cells. The echocardiogram of the hearts implanted with the Kir2.1-expressing heart muscle cells was altogether more normal than that of the others.

This paper is a significant contribution to the use of ESC-derived cells to treat heart patients. The induction of heart arrhythmias by ESC-derived heart muscle cells is a documented risk of their use. Li and his colleagues have effectively eliminated that risk in this paper by forcing the expression of a potassium rectifier channel in the ESC-derived heart muscle cells. Also, because these cells were completely differentiated and did not have any interloping pluripotent cells in their culture, tumor formation was not observed.

There are a few caveats I would like to point out. First of all, the increase in survival rate above the control is not that impressive. The improvement in heart function parameters is certainly encouraging, but because the survival rates are not that higher than the control mice that received no treatment, it appears that these benefits were only conferred to those mice who survived in the first place.

Secondly, even though the heart attacks were induced in the ventricles of the heart, Li and his colleagues injected a mixture of heart muscle cells that included atrial, ventricular, nodal and heart fibroblasts. This provides an opportunity for beat mismatches and a “substrate for ventricular tachycardia” as Li puts it. In the future, the transplantation of just ventricular heart muscle cells would be cleaner experiment. Since these mice were not observed long enough to observe potential arrythmias that might have arisen from the presence of a mixed population in the ventricle.

Finally, in adapting this to humans might be difficult, since the hearts of mice beat so much faster than those of humans. It is possible that even if human cardiomyocytes were engineered with Kir2.1-type channels, that arrythmias might still be a potential problem.

All of us have probably heard of Crohn’s disease or have probably known someone with Crohn’s disease. While the severity of this disease varies from patient to patient, some people with Crohn’s disease simply cannot get a break.

Crohn’s disease is one of a group of diseases known as IBDs or “Inflammatory Bowel Diseases.” IBDs include Crohn;s disease, which can affect either the small or large intestine and rarely the esophagus and mouth, ulcerative colitis, which is restricted to the large intestine, and other rarer types of IBDs known that include Collagenous colitis, Lymphocytic colitis, Ischaemic colitis, Diversion colitis, Behçet’s disease, and Indeterminate colitis.

Crohn’s disease (CD) involves the patient’s immune system attacking the tissues of the gastrointestinal tract, which leads to chronic inflammation within the bowel. While the exact mechanism by which this disease works is still not completely understood and robustly debated, Crohn’s disease was originally thought to be an autoimmune disease in which the immune system recognizes some kind of surface protein in the gastrointestinal tract as foreign and then attacks it. However, genetic studies of CD, linked with clinical and immunological studies have shown that this is not the case. Instead, CD seems to be due to a poor innate immunity so that the bowel has an accumulation of intestinal contents that breach the lining of the gastrointestinal tract, resulting in chronic inflammation. A seminal paper by Daniel Marks and others in the Lancet in 2006 provided hard evidence that this is the case. When Marks and others tested the white blood cells from CD patients and their ability to react to foreign invaders, those cells were sluggish and relatively ineffective. Therefore, Crohn’s seems to be an overactivity of the acquired immunity to make up for poor innate immunity.

Given all that, one of the biggest, most painful consequences of CD are anal fistulas. If those sound painful it’s because they are. A fistula is a connection between to linings in your body that should not normally be connected. In CD patients, the anus and the attached rectum get kicked about by excessive inflammation and tears occur. These tears heal, but the healing can cause connections between linings that previously did not exist. Therefore fecal material not comes out of the body in more than one place. Sounds disgusting? It gets worse. Those areas that leak feces are not subject to extensive pus formation and they must be fixed surgically. But how do you fix something that is constantly inflamed? It’s an ongoing problem in medicine.

Enter stem cells to the rescue, maybe. In Spain, a multicenter clinical study has just been published that shows that fat-derived mesenchymal stem cells might provide a better way to treat these fistulas in CD patients. Mesenchymal stem cells have the ability to suppress inflammation, and for that reason, they are excellent candidates to accelerate healing in cases such as these.

Galindo and his group took 24 CD patients who had at least one draining fistula (yes, some have more than one) and gave them 20 million fat-derived mesenchymal stem cells. These cells were extracted from someone else, which is an important fact, since liposuction procedures on these patients might have added to their already surfeit of inflammation.

For this treatment, the cells were administered directly on the lesion, which is almost certainly important. If the closing of the fistula was incomplete after 12 weeks, then the patients were given another dose of 40 million fat-derived mesenchymal stem cells right on the lesion. All these patients were followed until week 24 after the initial stem cell administration.

The results were very hopeful. There were no major adverse effects six months after the stem cell treatment. This is a result seen over and over with mesenchymal stem cells – they are pretty safe when administered properly. Secondly, full analysis the data showed that at week 24 69.2% of the patients showed a reduction in the number of draining fistulas. Even more remarkably, 56.3% of the patients achieved complete closure of the treated fistula. That is just over half. Also, 30% of the cases showed complete closure of all existing fistulas. These results are exciting when you consider the criteria they used for complete closure: absence of draining pus through its former opening. complete “re-epithelization” of the tissue, which means that the lining of the tissue is healed, looks normal and is properly attached to the proper neighbors, and magnetic resonance image (MRI) scans of the region must look normal. For these patients, the MRI “Score of Severity,” which is a measure of the structural abnormality of the anal region, showed statistically significant reductions at week 12 with a marked reduction at week 24. Folks that’s good news.

Galindo interprets his results cautiously and notes that this is a small study, which is true. He also states that the goal of this study was to ascertain the safety of this technique, and when it comes to safety, this technique is certainly safe. When it comes to efficacy, another larger study is required that specifically examined the efficacy of this technique. Galindo is, of course, quite correct, but this is certainly a very exciting result, and hopefully these cells will get further chances to “strut their therapeutic stuff.”

After a heart attack, inflammation in the heart kills off heart muscle cells and fibroblasts in the heart make a protein called collagen, which forms a heart scar. The heart scar does not contract and does not conduct electrochemical signals. The scar will contract over time, but its presence can lead to abnormal heart rhythms, also known as arrhythmias. Arrythmias can be fatal, since they can cause a heart attack. To prevent a heart attack, physicians will treat heart attack patients with a group of drugs called beta-blockers that slow down the heart rate and protect the heart from the deleterious effects of norepinephrine (secreted by the sympathetic nerve inputs to the heart). An alternative treatment is digoxin or digitalis, which is a chemical found in foxglove. Digitalis inhibits ion pumps in heart muscle cells and slows the heart and the force of its contractions. Digitalis, however, interacts with a whole shoe box fill of drugs, has a very long half-life, and is hard to dose. Therefore it is not the first choice.

Given all this, helping the heart to make a smaller heart scar is a better strategy for treating a heart after a heart attack. To accomplish this, you need to inhibit the heart fibroblasts that make the heart scar in the first place. Secondly, you must move something into the place of the dead cells. Otherwise, the heart could burst or scar tissue will move into the area anyway.

To that end, Yigang Wang and his colleagues at the University of Cincinnati Medical Center in Ohio have published an ingenious paper in which they tried two different strategies to reduce the size of the heart scar, which concomitantly increased the colonization of the heart by induced pluripotent stem cells engineered to express a sodium-calcium exchange pump.

Previously, Wang and his colleagues used a patch to heal the heart after a heart attack. The patch consisted of endothelial cells, which make blood vessels, induced pluripotent stem cells engineered to make a sodium-calcium exchange pump called NCX1, and embryonic fibroblasts. This so-called tri-cell patch makes new blood vessels, establishes new heart muscle, and the foundational matrix molecules to form a platform for beating heart muscle.

In order to get these cells to spread throughout the injured heart, Wang and others used a reagent that specifically inhibits heart fibroblasts. They used a small non-coding RNA molecule. A group of microRNAs called miR-29 family are downregulated after a heart attack. As it turns out, these microRNAs inhibit a group of genes that involved in collagen deposition. Therefore, by overexpressing miR-29 microRNAs, they could prevent collagen deposition and reduce scar formation.

The experimental design in this paper is rather complex. Therefore, I will go through it slowly. First, they tried to overexpress miR-29 microRNAs in cultured heart fibroblasts and sure enough, they inhibited collagen synthesis. Cells overexpressing miR-29 made less than a third of the collagen of their normal counterparts. When they placed these fibroblasts into the heart and induced heart attacks, again, they made significantly less collagen when they were expressing miR-29.

Then they used their miR-29 RNAs by injecting them directly into the heart before inducing a heart attack, and then after the heart attack, they applied the tri-patch. Their results were significant. The scar size was smaller (almost one-third the size of the controls), and the density of blood vessels was much higher in the tri-patched hearts treated with miR-29. The induced pluripotent stem cells differentiated into heart muscle cells and spread throughout the heart. Heart function measures also consistently went up too. The echiocardiograph before more normal, the ejection fraction went up, the % shortening of the heart muscle fibers was increased, and the relaxation phase of the heart (diastole) also was not so puffy (see graphs and figures below).

There is a cautionary note to this study. Inhibiting collagen formation after a heart attack could create soft fragile regions of the heart that are subject to rupture should the vascular systolic pressure increase. While that threat was not observed in this study, human hearts, which are much larger, would be much more susceptible to such a mishap. Therefore, while this study is interesting and suggest a strategy in humans, it requires more testing and refinement before anyone can even think about applying it to humans.

Down syndrome (DS) results when human babies have three copies of chromosome 21 rather than the normal two copies. However, three copies of pieces of chromosome 21 can also cause DS, and the region of chromosome 21 called the “Down Syndrome Critical Region” can also cause the symptoms of DS. The Down Syndrome Critical Region is located 21q21–21q22.3. Within this region are several genes, that, when present in three copies, seem to be responsible for the symptoms of DS. These genes are APP or amyloid beta4 precursor protein, SOD1 or Superoxide dismutase, DYRK or Tyrosine Phosphorylation-Regulated Kinase 1A, IFNAR or Interferon, Alpha, Beta, and Omega, Receptor, DSCR1 or the Down Syndrome Critical Region Gene 1 (some sort of signaling protein), COL6A1 or Collagen, type I, alpha 1, ETS2 or Avian Erythroblastosis Virus E26 Oncogene Homolog 2, and CRYAz or alpha crystalline (a protein that makes the lens of the eye).

All of these genes have been studied in laboratory animals, and the overproduction of each one of them can produce some of the symptoms of DS. For example, APP overproduction in mice leads to the death of neurons in the brain and inadequate transport of growth factors in the brain (see A.Salehi et al., Neuron, July 6, 2006; and S.G. Dorsey et al., Neuron, July 6, 2006). Also, the overexpression of CRYA1 seems to cause the increased propensity of DS patients to suffer from cataracts. Likewise, overexpression of ETS2 leads to the head and facial abnormalities in mice that are normally seen in human DS patients (Sumarsono SH, et al. (1996). Nature 379 (6565): 534–537).

People can also have only portions of the DS Critical Region triplicated and this leads to graded types of DS that only have some but not all of the symptoms of DS.

Why all this introduction to DS? It is among the most frequent genetic causes of intellectual disability. Therefore, finding a way to improve the cognitive abilities of DS patients is a major goal. T

There is a mouse strain called Ts65Dn mice that recapitulates some major brain structural and behavioral symptoms of DS and these include reduced size and cellularity of the cerebellum and learning deficits associated with the hippocampus.

Roger Reeves at Johns Hopkins University has used a drug that activates the hedgehog signaling pathway to reverse the brain deficits of Ts65Dn mice. Yes you read that right.

A single treatment given the newborn mice of the Sonic hedgehog pathway agonist SAG 1.1 (SAG) results in normal cerebellar morphology in adults.

But wait, there’s more. SAG treatment at birth also improved the hippocampal structure and function. The hippocampus is involved in learning and memory.

SAG treatment resulted in behavioral improvements and normalized performance in a test called the “Morris water maze task for learning and memory. The Morris water maze test essentially takes a mouse from a platform in shallow water and then moves the mouse through the maze and then leave it there. The mouse has to remember how they got there and retrace their steps to get back to the platform before they get too tired from all that swimming. Normally Ts65Dn mice do very poorly at this test. However, after treating newborn Ts65Dn mice with SAG, they improved their ability to find their way back.

SAG treatment also produced other effects in the brain. For example, the ratios of different types of receptors in the brain associated with memory are skewed in Ts65Dn mice, but after treatment with SAG, these ratios became far more normal. Also, the physiology of learning and memory was also more normal in the brains of SAG-treated Ts65Dn mice.

These results are extremely exciting. They confirm an important role for the hedgehog pathway in cerebellar development. Also, they suggest that the development of the cerebellum (a small lobe at the back of the brain involved in coordination and fine motor skills, direct influences the development of the hippocampus. These results also suggest that it might be possible to provide a viable therapeutic intervention to improve cognitive function for DS patients.

This excitement must be tempered. This is an animal model and not a perfect animal model. Also, it is unclear if such a compound will work in humans. Much more work must be done, but this is a fascinating start.

Johnny Huard and his co-workers from the McGowan Institute for Regenerative Medicine at the University of Pittsburgh have isolated a slowly-adherent stem cell population from skeletal muscle called muscle-derived stem cells or MDSCs (see Deasy et al Blood Cells Mol Dis 2001 27: 924-933). These stem cells can form bone and cartilage tissue in culture when induced properly, but more importantly when MDSCs are engineered to express the growth factor Bone Morphogen Protein-2 (BMP-2), they make better bone and do a better job of healing bone lesions than other engineered muscle-derived cells (Gates et al., J Am Acad Orthop Surg 2008 16: 68-76).

In most experiments, MDSCs are infected with genetically engineered viruses to deliver the BMP-2 genes, but the use of viruses is not preferred if such a technique is to come to the clinic. Viruses elicit and immune response and can also introduce mutations into stem cells. Therefore a new way to introduce BMP-2 into stem cells is preferable.

To that end, Huard and his colleagues devised an ingenious technique to feed BMP-2 to implanted MDSCs without using viruses. They utilized a particle composed of heparin (a component of blood vessels) and a synthetic molecule called poly(ethylene arginylaspartate diglyceride), which is mercifully abbreviated PEAD. The PEAD-heparin delivery system formed a so-called “coacervate,” which is a tiny spherical droplet that is held together by internal forces and composed of organic molecules. These PEAD-heparin coacervates could be loaded with BMP-2 protein and they released slowly and steadily to provide the proper stimulus to the MDSCs to form bone.

When tested in culture dishes, the BMP-2-loaded coacervates more than tripled the amount of bone made by the MDSCs, but when they were implanted in living rodents the presence of the BMP-2-loaded coacervates quadrupled the amount of bone made by the MDSCs.

This technique provides a way to continuously deliver BMP-2 to MDSCs without using viral vectors to infect them. These carriers do inhibit the growth or function of the MDSCs and activate their production of bone.

This paper used a “heterotropic bone formation assay” which is to say that cells were injected into the middle of muscle and they formed ectopic bone. The real test is to see if these cells can repair actual bone lesions with this system.

Induced pluripotent stem cells are made from adult cells by means of genetic engineering techniques that introduce into the cells a combination for four different genes that drive the cells to de-differentiate into a cell that has many of the characteristics of embryonic stem cells without the destruction of embryos.

A new study from the laboratory of Juergen Knoblich at the Institute of Molecular Biotechnology in Vienna has mixed induced pluripotent stem cells (iPSCs) to form structures of the human brain. He largely left the cells alone to allow them to form the brain tissue, but he also placed them in a spinning bioreactor that constantly circulates the culture medium and provides nutrients and oxygen to the cells. One other growth factor he supplied to the cells was retinoic acid, which is made by the meninges that surround our brains. All of this and the cells not only divided, differentiated and assembled, but they formed brain structures that had all the connections of a normal brain. These brain-like chunks of tissue are called “mini-brains” and the recent edition of the journal Nature reports their creation.

“It’s a seminal study to making a brain in a dish,” says Clive Svendsen, a neurobiologist at the University of California, Los Angeles. Svendsen was not involved in this study, but wishes he was. Of this study, Svendsen exclaimed, “That’s phenomenal” A fully formed artificial brain is still years and years away, but the pea-sized neural clumps developed in Knoblich’s laboratory could prove useful for researching human neurological diseases.

Researchers have previously used pluripotent human stem cells to grow structures that resemble the developing eye (Eiraku, M. et al. Nature 472, 51–56 (2011), and even tissue layers similar to the cerebral cortex of the brain (Eiraku, M. et al. Cell Stem Cell 3, 519–532 (2008). However, this latest advance has seen bigger and more complex neural-tissue clumps by first growing the stem cells on a synthetic gel that resembled natural connective tissues found in the brain and elsewhere in the body. After growing them on the synthetic gel, Knoblich and his colleagues transferred the cells to a spinning bioreactor that infuses the cells with nutrients and oxygen.

“The big surprise was that it worked,” said Knoblich. The clump formed structures that resembled the brains of fetuses in the ninth week of development.

Under a microscope, the blobs contained discrete brain regions that seemed to interact with one another. However, the overall arrangement of the different proto-brain areas varied randomly across tissue samples. These structures were not recognizable physiological structures.

A cross-section of a brain-like clump of neural cells derived from human stem cells.

“The entire structure is not like one brain,” says Knoblich, who added that normal brain maturation in an intact embryo is probably guided by growth signals from other parts of the body. The tissue balls also lacked blood vessels, which could be one reason that their size was limited to 3–4 millimeters in diameter, even after growing for 10 months or more.

Despite these limitations, Knoblich and his collaborators used this system to model key aspects of microcephaly, which is a condition that causes extremely stunted brain growth and cognitive impairment. Microcephaly and other neurodevelopmental disorders are difficult to replicate in rodents because the brains of rodents develop differently than those of humans.

Knoblich and others found that tissue chunks cultured from stem cells derived from the skin of a single human with microcephaly did not grow as large as clumps grown from stem cells derived from a healthy person. When they traced this effect, they discovered that it was due to the premature differentiation of neural stem cells inside the microcephalic tissue chunks, which depleted the population of progenitor cells that fuels normal brain growth.

The findings largely confirm prevailing theories about microcephaly, says Arnold Kriegstein, a developmental neurobiologist at the University of California, San Francisco. But, he adds, the study also demonstrates the potential for using human-stem-cell-derived tissues to model other disorders, if cell growth can be controlled more reliably.

“This whole approach is really in its early stages,” says Kriegstein. “The jury may still be out in terms of how robust this is.”